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Detailed Analysis of the Surface Area and Elasticity in the Saturated 1,2-Diacylphosphatidylcholine/Cholesterol Binary Monolayer System Tsubasa Miyoshi* and Satoru Kato Department of Physics, Graduate School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan

Downloaded by UNIV OF MICHIGAN ANN ARBOR on September 7, 2015 | http://pubs.acs.org Publication Date (Web): August 14, 2015 | doi: 10.1021/acs.langmuir.5b01775

S Supporting Information *

ABSTRACT: The surface pressure−area (π−A) isotherms of DMPC, DPPC, and DSPC/cholesterol binary monolayers were systematically measured with great care to gain insight into the lateral molecular packing in these binary monolayer systems. The average molecular area A and the area elastic modulus Cs−1 at a given surface pressure were calculated as a function of cholesterol mole fraction xchol. As a result, data reliable enough for the analysis of detailed phase behavior were obtained. We identified several characteristic phase regions and assigned the phase state in each region on the basis of the deviation of A(xchol) and Cs−1(xchol) from ideal additivity. We also estimated the partial molecular areas of DMPC, DPPC, DSPC, and cholesterol in the single-phase regions, where Cs−1(xchol) values fell on an ideal additivity curve. We found that the addition of cholesterol induces the formation of a highly condensed phase where the diacylphosphatidylcholine (diacyl PC) molecule has a surface area even smaller than that in the solid phase, irrespective of the surface pressure and the chain length of diacyl PC. Here, we call the cholesterol-induced condensed phase the CC phase. Furthermore, we demonstrated that the basic features of A(xchol) and Cs−1(xchol) profiles can be explained semiquantitatively by assuming the state of vicinity lipids surrounding sparsely distributed cholesterol molecules in the low xchol region as a third state of the diacyl PC molecule in addition to the states in the pure diacyl PC monolayer and in the CC phase.

1. INTRODUCTION The lipid packing structures in biomembranes are thought to be closely related to their biological functions. Cholesterol is widely distributed in biomembranes and is suggested to control the biomembrane physical properties through the modification of the lipid molecular packing structures and to play a key role in the functional microdomain called a lipid raft.1−10 In order to elucidate the effects of cholesterol on the lateral molecular packing in a lipid membrane, the monolayer formed at the air/water interface has been intensively and extensively studied as a useful system to acquire microscopic information from macroscopic quantities. The most widely used method for the analysis of the monolayer system is the surface pressure− area (π−A) isotherm measurement.11−34 The π−A isotherm of the well-characterized dipalmitoylphosphatidylcholine (DPPC) monolayer shows a broad plateau with nearly constant surface pressure12 due to the transition from the liquid-expanded (LE) phase to the liquid-condensed (LC) phase. Intermolecular interaction in cholesterol-containing monolayers can be evaluated by analyzing the cholesterol concentration dependence of the average molecular area, elasticity, and excess Gibbs free energy of mixing at fixed surface pressures.14,16,17,22,24,27,29−31,34 The addition of cholesterol to the DPPC monolayer induces the formation of a more tightly packed state than the LE and LC phases as the average molecular area deviates negatively from that of ideal mixing.14,16,17,22,24,27,31,34 Thus, cholesterol is known to have © 2015 American Chemical Society

a condensing effect on the diacylphosphatidylcholine (diacyl PC) monolayers. However, there has been no detailed analysis of the phase behavior in the mixed monolayer based on the π− A isotherm measurements because their reproducibility is easily affected by various factors such as temperature, dust contamination, and the experimenter’s skill. The manner of phase separation in a monolayer is directly observed by fluorescence microcopy,13,35−41 Brewster angle microscopy18,20,21,26,27,29,30,33,42−44 and atomic force microscopy of the monolayer transferred onto a substrate were conducted.15,37,45−49 In the DPPC/cholesterol monolayers at 10 mN/m the microscopic images at cholesterol mole fraction xchol = 0.05 suggested phase separation whereas those at xchol = 0.3 were fairly homogeneous, indicating the formation of a single phase.47 Although these observations are useful in determining the phase state of the monolayer, they are far from systematic, and the data points are too few to determine the detailed phase behavior. In this study, we repeated the π−A isotherm measurements until at least five profiles converged within tolerable error for the detailed analysis of the phase behavior of the saturated diacyl PC/cholesterol mixed monolayer system. As a result, we found that several phase regions can be discernible on the basis Received: May 15, 2015 Revised: July 14, 2015 Published: August 8, 2015 9086

DOI: 10.1021/acs.langmuir.5b01775 Langmuir 2015, 31, 9086−9096

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xchol, and the obtained A(xchol) in a single-phase region was fitted to a straight line (eq 1), where the partial molecular areas, ALipid and Achol, are assumed to be kept constant. The value of Ai(0) corresponds to the partial molecular area of diacyl PC in the single phase and the value of Ai(1) corresponds to that of cholesterol.

of the cholesterol concentration dependence of the average molecular area and elasticity and that the assumption of at least three states for each molecule is needed to explain our data.


2. MATERIALS AND METHODS 2.1. Materials. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC; synthetic, purity >99%), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC; synthetic, purity >99%), 1,2-distearoyl-sn-glycero3-phosphocholine (DSPC; synthetic, purity >99%), and cholesterol (ovine wool, purity >98%) were purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification. These diacylphosphatidylcholines (diacyl PCs) are saturated and have a phosphocholine as a hydrophilic head but differ in the hydrocarbon chain length; the numbers of carbon atoms in the acyl chains are 14 (DMPC), 16 (DPPC), and 18 (DSPC). A series of samples for π−A isotherm measurements of a diacyl PC/ cholesterol monolayer system were prepared from the same stock solutions to minimize the fluctuation of the relative concentration of cholesterol. The stock solutions of diacyl PCs and cholesterol were prepared with chloroform/methanol (4:1, v/v) and stored at −20 °C until use because it took 7−10 days to finish the series of measurements for one binary monolayer system (the number of measurements was on the order of 100). To reduce pipetting error, more than 100 μL of diacyl PC stock solution and more than 10 μL of cholesterol stock solution were mixed to prepare a sample solution. It was confirmed that there was little difference between the results in the first and final days. 2.2. Surface Pressure−Area (π−A) Isotherm Measurement. The π−A isotherms of diacyl PC/cholesterol binary monolayers were measured using a computer-controlled Langmuir-type film balance (USI system, Fukuoka, Japan) with more cholesterol concentrations than in previous works. Before measurement, the trough (100 × 290 mm2) was carefully cleaned several times with ethanol and distilled− deionized water (Milli-Q, Milipore Co., Milford, MA) and filled with distilled−deionized water as the aqueous subphase. The diacyl PC solution (30 μL, 1 mg/mL) was spread over the subphase between barriers. After the solvent was evaporated for about 10 min, the monolayer was compressed at a rate of 0.1 mm/s. The subphase temperature was kept at 25.0 ± 0.1 °C. In order to obtain experimental data with high accuracy, the measurements under the same conditions were repeated until at least five π−A isotherm profiles converged so that the average molecular area at 30 mN/m calculated from these data had an error within 0.01 nm2. 2.3. Analysis of π−A Isotherms. The average molecular area A and the area elastic modulus Cs−1 at a fixed surface pressure were calculated as a function of cholesterol mole fraction xchol from a series of π−A isotherms according to the standard analysis procedure.16,17,25,27,32,50,51 The ideal average molecular area Ai(xchol) was calculated from

Ai (xchol) = (1 − xchol)ALipid + xcholAchol

3. RESULTS 3.1. Detailed Surface Area Behavior of the DPPC/ Cholesterol Binary Monolayer System. The π−A isotherms of DPPC, cholesterol, and DPPC/cholesterol binary monolayers were carefully measured with more cholesterol concentrations than in previous studies13−17,20,22−24,28 to obtain detailed information on the molecular interactions in the monolayer formed on an aqueous subphase (Figure 1). With

Figure 1. π−A isotherms of DPPC, cholesterol, and DPPC/ cholesterol binary monolayers on an aqueous subphase at 25 °C. The xchol values are indicated. The π−A isotherm profile shifts toward lower A as xchol increases.

increasing cholesterol mole fraction xchol, the average molecular area A range where the liquid-expanded (LE) and liquidcondensed (LC) phases coexist became narrower and the transition surface pressure increased slightly. On the basis of Figure 1, the values of A were plotted as a function of xchol at a fixed surface pressure (π = 5, 10, 15, 20, 25, 30, and 35 mN/m) to analyze the intermolecular interaction and the extent of the cholesterol-induced condensing effect (Figure 2). The average molecular area in the ideally mixed DPPC/cholesterol binary monolayer Ai was calculated by eq 1 for each surface pressure (dotted lines in Figure 2). If DPPC and cholesterol molecules are mixed ideally or are completely phase-separated, then the A(xchol) profiles should be placed on the dotted line calculated on the basis of the additivity of their intrinsic areas. In fact, however, there must be some interaction between DPPC and cholesterol molecules, which results in deviation from the additivity line regardless of xchol, indicating attractive interaction (condensing effect). The magnitude of the condensing effect was much larger at π ≤ 10 mN/m than at π ≥ 15 mN/m, suggesting that on adding cholesterol the molecular area of DPPC in the LE phase decreases to a greater extent than that in

(1)

where ALipid and Achol are the molecular areas in the pure diacyl PC and cholesterol monolayer, respectively. The area compressibility modulus Cs(xchol) was calculated from

Cs(xchol) = −

1 ⎛⎜ dA ⎞⎟ A ⎝ dπ ⎠

(2)

The ideal compressibility modulus Csi(xchol) was calculated from

Csi(xchol) = − CLs

1 {(1 − xchol)CsLALipid + xcholCscAchol } Ai (xchol)

(3)

Ccs

and are the compressibility moduli of the pure diacyl PC where monolayer and the pure cholesterol monolayer, respectively. The area elastic modulus Cs−1 was calculated as the inverse of the Cs values. The partial molecular areas of diacyl PC and cholesterol in a single phase were estimated according to the method described by Edholm et al.52 Briefly, the average molecular area A was plotted as a function of 9087


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on an ideal curve calculated by assuming that the elastic modulus of cholesterol in the binary monolayer is just the same as that in the pure cholesterol monolayer (eq 3). We designated the latter region at xchol = 0.28−0.5 as the CC phase in the previous section. Here, we designate the former region at xchol= 0.09−0.2 as the LC* phase for convenience (Discussion section). The conspicuous characteristic of the LC* phase is its smaller elastic modulus compared to that of the LC phase. If these regions in addition to the LC phase at xchol = 0 represent single phases, then the regions between these phases (xchol = 0−0.09 and 0.2−0.28) must be two-phase coexistence regions. At xchol> 0.5 it was difficult to determine the phase boundaries based on the Cs−1(xchol) profile due to scattering of the data, which may be caused by uncontrollable segregation of pure cholesterol domains, although the A(xchol) profile had a clear breakpoint at xchol ≈ 0.6. Therefore, we focused our analysis on the lower xchol region. Thus, we identified at least five regions based on the Cs−1(xchol) profile. Roughly speaking, three of the four boundaries among these five regions (xchol = 0.09, 0.28, and 0.5) have their counterparts in the A(xchol) profile (xchol = 0.12, 0.25, and 0.6, arrows in Figure 3A(a)). Although the agreement of the boundary positions was not perfect, the differences may be within experimental error considering that the boundary positions were selected at the positions of experimental data and the differences correspond to those between neighboring data points. The other boundary at xchol = 0.2 (arrow in Figure 3(b)) seemed to have no counterpart in the A(xchol) profile. At a low surface pressure of 10 mN/m, the Cs−1(xchol) profile seemed to have only one transition region at xchol = 0.25−0.6 (Figure 3B(b)). The regions at xchol < 0.25 and xchol> 0.6 seemed to be single phases, judging from the deviation from the ideal curves. The latter region (xchol > 0.6) corresponds to the CC phase described in the previous section though there may be another region in the vicinity of xchol = 1.0, where the CC phase and the pure cholesterol domain coexist. Thus, at least three regions were discerned in the Cs−1(xchol) profile. On the other hand, the A(xchol) profile was rather smooth, and the breakpoints in the A(xchol) profile did not seem to be coincide with the phase boundaries identified in the Cs−1(xchol) profile, suggesting that the phase behavior at low xchol may be more complex than expected from the Cs−1(xchol) profile. However, we did not further analyze the A(xchol) profile as it was difficult to unequivocally determine the breakpoint positions. 3.3. Diacylphosphatidylcholine Chain Length Dependence of Surface Area Behaviors. Figure 4 shows π− A isotherms of DMPC, DPPC, and DSPC monolayers. In spite of the hydrocarbon chain length difference in these diacyl PCs, the π−A isotherm profiles in the LE, LC, and solid (S) phases53,54 agreed well with each other. Whereas the DPPC monolayer assumed the S phase in addition to the LE and LC phases described in section 3.1, the DMPC monolayer assumed only the LE phase. In the DSPC monolayer the π−A isotherm profile seemed to have a small kink at π ≈ 55 mN/m in addition to the LC to S phase transition at π ≈ 60 mN/m.55,56 We did not further examine the characteristics of this kink because it is out of the scope of this article. Further study is needed to clarify them. The collapse pressure depended on the chain length; that is, the longer the chain length, the higher the collapse pressure. In order to evaluate the effect of the hydrocarbon chain length, π−A isotherms of DMPC/cholesterol and DSPC/ cholesterol binary monolayers were measured (Figure 5), and

Figure 2. Average molecular area analysis in DPPC/cholesterol binary monolayers at 5, 10, 15, 20, 25, 30, and 35 mN/m. Dotted lines indicate the ideal average molecular area Ai(xchol) at each surface pressure.

the LC phase (Figure 2). These overall characteristics in the π− A isotherms of the DPPC/cholesterol binary system are fundamentally consistent with those of previous studies13−17,23,24 Since we measured with more cholesterol concentrations than in previous works, Figure 2 gives further detailed information on the phase behavior in the binary system. Every A(xchol) profile in Figure 2 has a clear breakpoint from the region with a sharp decrease at lower xchol toward the region with a gentle slope (e.g., at xchol ≈ 0.5 (5 mN/m), 0.3 (15 mN/ m), and 0.15 (35 mN/m)), though the breakpoint shifted toward lower xchol values with increasing surface pressure. On the higher xchol side of the breakpoint the binary monolayer must be in a definable phase irrespective of surface pressure (see below). Here, we call it the cholesterol-induced condensed (CC) phase, as DPPC molecules must be in a highly ordered state, judging from the smaller partial molecular area of DPPC than in the LC phase (Discussion section). 3.2. Analysis of the Area Elastic Modulus in the DPPC/ Cholesterol Binary Monolayer System. In order to clarify the detailed phase behavior of the DPPC/cholesterol binary monolayer system, we calculated the area elastic modulus Cs−1 as a function of xchol in addition to A(xchol) according to the conventional analytical method (Materials and Methods section) and tried to identify as precisely as possible the boundaries of phase regions by comparing these data. Figure 3 shows examples of the Cs−1(xchol) and A(xchol) profiles at high and low surface pressures. The phase boundaries were determined by primarily using the Cs−1(xchol) profiles because the Cs−1 values in a phase-transition (twophase coexistence) region clearly deviated from the curve of ideal mixing of two molecules with appropriate constant molecular areas (dotted lines in Figure 3(b)). At a high surface pressure of 30 mN/m, the Cs−1(xchol) profile had at least two regions (xchol = 0.09−0.2 and 0.28−0.5) where Cs−1 values fell 9088


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Figure 3. (a) Average molecular area and (b) area elastic modulus in the DPPC/cholesterol binary monolayer system at (A) 30 mN/m and (B) 10 mN/m (a) as a function of xchol. The ideal average molecular area Ai(xchol) is plotted as a straight line according to eq 1 (dotted line in (a)). The black dotted curve in (b) shows the ideal area elastic modulus Csi(xchol) calculated on the basis of ideal mixing of pure cholesterol and DPPC. The other dotted curves in (b) were the Csi(xchol) profiles obtained by setting appropriate values for CLs in eq 3 under the condition of fixed Ccs . The regions where measured data fall on an ideal curve (arrows) were interpreted as a single phase.

the xchol dependences of the average molecular area A(xchol) and the area elastic modulus Cs−1(xchol) were analyzed. We focused on the phase behavior of these monolayers at 30 mN/m for comparison. Figure 6 shows the results of the analysis at 30 mN/m. The manners of deviation from ideal additivity in the DMPC/ cholesterol and DSCP/cholesterol monolayer systems were similar to those in the DPPC/cholesterol monolayer system at 10 and 30 mN/m, respectively, suggesting that the phase in the absence of cholesterol mainly rules the phase states induced by the addition of cholesterol. However, a close look at the data revealed that there were significant differences: In the DMPC/ cholesterol monolayer system the Cs−1(xchol) profile showed a two-step change in the xchol range from 0.2 to 0.7 (Figure 6A(b)). The terrace in the middle of the two-step change appeared at π ≥ 15 mN/m and became clearer as the surface pressure increased. The terrace may represent the CC phase because the elastic modulus of the DMPC molecule estimated by fitting the data in the plateau to an ideal curve was reasonable as the CC phase. Therefore, there might be another phase in the high xchol region. In addition, the A(xchol) profile showed clear breakpoints at xchol ≈ 0.2, 0.3, and 0.7, which

roughly corresponded to the phase boundaries identified in the Cs−1(xchol) profile, though there seemed to be no breakpoint at xchol ≈ 0.5 corresponding to the higher end of the terrace region in the Cs−1(xchol) profile. In the DSPC/cholesterol monolayer system an evident difference from the data in the DPPC/cholesterol monolayer system at 30 mN/m was that the Cs−1(xchol) data in the xchol range from 0 to 0.1 fell on the ideal curve despite the negative deviation of A(xchol) from the ideal line (Figure 6B). Another difference was that there seemed to be no definite region corresponding to the LC* phase seen in Figure 3A(b). The partial molecular areas of diacyl PC and cholesterol at 30 mN/m were calculated as described in section 2.3 and plotted as a function of the hydrocarbon chain length (Figure 7). The partial molecular areas of DPPC and DSPC in the LC phase, ALC Lipid, were almost the same as shown in Figure 4. In contrast, the partial molecular area in the CC phase, ACC Lipid, increased as the hydrocarbon chain length was decreased and became closer to ALC Lipid (Figure 7A). Anyway, the lateral packing of diacyl PC molecules is tighter in the CC phase than in the LC phase. On the other hand, the partial molecular area of cholesterol in the CC phase, ACC chol, was slightly larger than that in the pure 9089


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Figure 4. Comparison of π−A isotherms of DMPC, DPPC, and DSPC monolayers on the aqueous subphase at 25 °C.

cholesterol monolayer, Apure chol (Figure 7B). In addition, we calculated the partial molecular area of DPPC in the LC* phase, ALC * , by fitting the A(xchol) data in the xchol range from DPPC 0.12 to 0.2 in Figure 3A(a) to a straight line. The ALC * value DPPC CC was located just in the middle between the ALC DPPC and ADPPC LC values (Figure 7A), and the Achol* value was almost the same as ACC chol (Figure 7B). Moreover, we calculated the partial molecular area of cholesterol from the A(xchol) data in the xchol range from 0 to the first breakpoint on adding cholesterol, which we denote as ALC chol for convenience, because it should contain information on the boundary lipids surrounding a cholesterol molecule in the LC phase as discussed by Edholm et al.52 Here, we call the boundary lipids the vicinity lipids and will propose a model in which all of the DPPC molecules in the LC* phase are in the state of the vicinity lipid (Discussion section). If the small value of ALC chol in Figure 7B is due to the areal shrinkage of the vicinity lipid from the bulk lipid in the LC phase, then the value of LC (Apure chol − Achol)/n corresponds to the molecular area difference between the vicinity lipid and the bulk lipid. (n is the number of vicinity lipids surrounding a cholesterol molecule.) Figure 5. π−A isotherms of (A) DMPC, cholesterol, and DMPC/ cholesterol monolayers and (B) DSPC, cholesterol, and DSPC/ cholesterol monolayers on an aqueous subphase at 25 °C. The values of xchol are indicated.

4. DISCUSSION 4.1. Molecular State in the Cholesterol-Induced Condensed (CC) Phase. The detailed behavior of surface pressure−area (π−A) isotherms of the saturated diacyl PC/ cholesterol binary monolayer system was scrutinized carefully by repeating the measurements under the same conditions so as to achieve good reproducibility (Figures 1 and 5). As a result, we found that the phase behavior in the binary monolayer system is more complicated than described in previous studies, though all of the diacyl PC/cholesterol systems examined shared the characteristics that the addition of cholesterol caused condensation and induced a laterally tightly packed phase (here we denoted it as the cholesterol-induced condensed (CC) phase). In what state are the diacyl PC molecules in the CC phase? In order to gain insight into the lateral molecular packing in the CC phase we plotted the estimated partial molecular area of

DPPC in the CC phase, ACC DPPC, as a function of surface pressure and compared it to the π−A isotherm of the pure DPPC monolayer (Figure 8). The ACC DPPC(π) profile seems to show an inconspicuous transition at ∼20 mN/m. However, we will discuss below the CC phase as a single phase independent of π as it was difficult to conclude unequivocally whether there is a phase transition. The ACC DPPC values were evidently smaller than the molecular area of pure DPPC in the LC and S phases; i.e., DPPC molecules in the CC phase are in a highly condensed state. It is likely that cholesterol molecules are intercalated between diacyl PC molecules with relatively large headgroups to reduce the 9090


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Figure 6. (a) Average molecular area and (b) area elastic modulus in (A) the DMPC/cholesterol monolayer system and (B) the DSPC/cholesterol monolayer system at 30 mN/m as a function of xchol. The dotted line in (a) and the dotted curve in (b) indicate the ideal average molecular area Ai(xchol) and the ideal area elastic modulus Csi−1(xchol) calculated on the basis of ideal mixing, respectively.

chain tilt with respect to the surface normal. Incidentally, the chain tilt in the LC phase was estimated to be 33 ± 3°,57 which corresponds to a 19% increase in the surface area, whereas the difference between the molecular areas of DPPC in the LC and CC phases represents about 14% (Figure 7A). If this molecular area difference is attributed only to the chain tilt, then the tilt angle of DPPC in the CC phase is roughly estimated to be about 16° (cos 16°/cos 33° ≈ 1.14). Anyway, it is likely that in the diacyl PC/cholesterol binary monolayers cholesterol works as a spacer to release the stress caused by the mismatch between the cross-sectional areas of the headgroup and the hydrocarbon chains.58 In contrast, the partial molecular area of cholesterol in the CC phase, ACC chol, is slightly larger than its molecular area in the pure cholesterol monolayer (Figure 7B). There are at least two possibilities to explain this behavior of ACC chol. The first possibility comes from the anisotropic molecular packing. Since the cholesterol molecule has a platelike structure, the molecular packing might be anisotropic and fairly tight in the monolayer of pure cholesterol. On the other hand, as cholesterol molecules in the CC phase are surrounded by diacyl PC molecules, they may rotate more isotropically and consequently have larger

surface areas than in the pure cholesterol monolayer. The second possibility comes from the formation of a hydrogen bond between phosphatidylcholine and cholesterol molecules in the CC phase. The hydrogen bond may restrict the free wobbling motion of the cholesterol molecule so as to increase its average tilt with respect to the surface normal. The increase in the tilt angle results in an increase in the surface area.59 4.2. Three-State Model to Explain the Phase States in the DPPC/Cholesterol Monolayers at 30 mN/m. Although the phase behaviors of the diacyl PC/cholesterol monolayer systems had common features, they were different in details, depending on the surface pressure and whether the monolayer phase in the absence of cholesterol was the LC phase or the LE phase. Here, we will first discuss the detailed phase behavior of the DPPC/cholesterol monolayer system at 30 mN/m as a typical example where the Cs−1(xchol) and A(xchol) profiles seemed to explicitly reflect changes in the phase state. We primarily adopted the Cs−1(xchol) profile and used the A(xchol) profile as supplementary data to determine the phase boundaries because the former profile exhibited clear changes at the phase boundaries. The phase boundaries were identified according to the following criteria: (1) We assume that the 9091


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Figure 8. Surface pressure dependence of the partial molecular area of CC DPPC in the CC phase, ACC DPPC(π) (○). The values of ADPPC were estimated from the xchol dependence of the average molecular area in the CC phase region at fixed surface pressures and were plotted together with the π−A isotherm of the pure DPPC monolayer.

these diacyl PC molecules under the influence of a single cholesterol molecules as vicinity lipids. 4.2.2. 0.1 ≲ xchol ≲ 0.2. In this region the xchol values were on a curve derived from the ideal mixing of cholesterol with an elastic modulus of ∼670 mN/m and DPPC with that of ∼115 mN/m, which was smaller than the value estimated in the pure DPPC monolayer (∼170 mN/m) and may correspond to the elastic modulus of the vicinity lipid. As for the A(xchol) profile, there is a clear breakpoint at xchol ≈ 0.12. We infer that the monolayer in this region is composed of only two kinds of molecules, i.e., cholesterol and the vicinity lipid (here we call the phase the LC* phase). If it is correct that the bulk DPPC molecules in the LC phase disappear at xchol ≈ 0.12, then the number of vicinity lipids surrounding one cholesterol molecule is calculated to be 7.3. 4.2.3. 0.2 ≲ xchol ≲ 0.3. In this region Cs−1(xchol) showed a sharp rise, suggesting the appearance of diacyl PC molecules with a high elastic modulus (∼215 mN/m). Therefore, we identified this region as the LC*−CC coexistence region. 4.2.4. 0.3 ≲ xchol ≲ 0.6. Since the Cs−1(xchol) profile in this region could be roughly fitted to an ideal curve, the binary monolayer may be in a single phase, which we denoted as the CC phase. We already discussed the characteristics of the CC phase on the basis of the partial molecular areas estimated from A(xchol) (section 4.1). 4.2.5. xchol ≳ 0.6. Although the Cs−1(xchol) values were scattered at xchol ≳ 0.6, A(xchol) showed a clear breakpoint at xchol ≈ 0.6 probably because of the formation of pure cholesterol domains.13 Considering the behavior of Cs−1(xchol), the phase behavior in this region may be more complicated than the coexistence of the CC phase and pure the cholesterol phase. In the above discussion we introduced three kinds of molecular states for a DPPC molecule: (1) the state in the pure DPPC monolayer in the LC phase (α1 state), (2) the state in the vicinity of lipids in the LC* phase (α2 state), and (3) the

Figure 7. Chain length dependence of the partial molecular areas of (A) diacyl PC ALipid and (B) cholesterol in the LC phase (●), the CC phase (○), and the LE phase (*) at 30 mN/m. ALipid (×) and Achol (★) in the LC* phase and Achol (−□−) in the pure cholesterol monolayer are also shown.

cholesterol molecule has a constant elastic modulus irrespective of the phase state. (2) The region where the Cs−1(xchol) values are on a curve derived from the ideal mixing of cholesterol and diacyl PC with an appropriate elastic modulus represents a single phase. (3) In the region other than (2), two phases or more than two kinds of molecules with different surface areas coexist. At least five regions were identified according to the above criteria (Figure 3A). Note that the value of xchol at the phase boundaries contains an error on the order of the xchol interval between neighboring experimental data. 4.2.1. xchol ≲ 0.1. The elastic modulus in this region decreased with increasing x chol as described previously13−17,24,28,34 and could not be fitted to an ideal curve. In addition, the average molecular area A decreased greatly with increasing xchol, and the partial molecular area of cholesterol was estimated to be 0.11 nm2, which is unreasonably smaller than the molecular area in the pure cholesterol monolayer (0.38 nm2) (Figure 7B). As suggested previously,52 the DPPC molecules surrounding the sparsely distributed cholesterol molecule may have a smaller surface area and a smaller elastic modulus than bulk ones in the LC phase. Here, we denote 9092


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Langmuir highly condensed state in the CC phase (α3 state). We speculate that the hydrocarbon chains of diacyl PC in the CC phase are in the all-trans state and their tilt with respect to the surface normal is reduced compared to that in the LC phase as discussed in the previous section and that the hydrocarbon chains in the vicinity lipid are not in the all-trans state, considering its low elastic modulus (Figure 10). We will show in the next section that the assumption of these three states can semiquantitatively explain the Cs−1(xchol) and A(xchol) profiles in the DPPC/cholesterol monolayer system at 30 mN/m except for the high xchol region. 4.3. Simulation Based on the Three-State Model. In the previous section, we introduced three states (α1, α2, and α3) for a DPPC molecule. We symmetrically assumed three states for a cholesterol molecule: (1) the state in the pure cholesterol monolayer (β1 state), (2) the state in the CC phase (β2 state), and (3) the state in the LC and LC* phase (β3 state). For simplicity, we did not assume the vicinity cholesterol surrounding a single diacyl PC molecule in the high xchol region. The molecule in each of these six states has two parameters, A and Cs−1. Fortunately, these 12 parameters were easily determined from the data in Figure 3A because we fixed the elastic modulus of cholesterol and identified the LC* and CC phase regions in the DPPC/cholesterol monolayer system at 30 mN/m. The parameters we determined are listed in Table S1 in the Supporting Information. Another factor we need to determine to simulate the experimental data is the distributions of these states as a function of xchol. We simply assumed the distributions of DPPC states as the α1 state is linearly replaced by the α2 state in section 4.2.1 described in the previous section and as the α2 state is linearly replaced by the α3 state in section 4.2.3 (Figure S1 in Supporting Information). The distributions of cholesterol states were assumed in a similar way. The average molecular area A and the elastic modulus Cs−1 were calculated by eqs S7 and S8 in the Supporting Information. Figure 9 shows the result of the simulation based on the above assumption, which agrees well with the experimental data except for the high xchol region. Thus, the three-state model can sufficiently explain the essential characteristics of the phase states in the DPPC/cholesterol monolayer system at 30 mN/m. Finally, it should be mentioned that the π−A isotherm of the pure DPPC monolayer did not show zero slope in the coexistence region of the LE/LC transition (Figures 1 and 4), which is obtained under uncommon conditions of an ultraclean system, pure materials, slow compression, and a stable film described by Hifeda and Rayfield. Further studies are needed to evaluate the effects of these conditions on the detailed phase behavior of the diacyl PC/cholesterol system. Moreover, further experiments such as Brewster angle microscopy, fluorescence microscopy, and grazing incidence X-ray diffraction should be performed to confirm our results because the information obtained from the π−A measurements is limited. 4.4. Phase States in DMPC/Cholesterol and DSPC/ Cholesterol Monolayers. The A(xchol) and Cs−1(xchol) profiles in the DSPC/cholesterol monolayer system at 30 mN/m showed similar characteristics to those in the DPPC/ cholesterol monolayer system (Figures 3A and 6B). As pointed out in the Results section, in the former system the Cs−1 data in the xchol range from 0 to 0.1 fell on an ideal curve, suggesting a single phase according to the criteria described in section 4.2. This is inconsistent with the sharp decrease in A, which

Figure 9. Simulation of (A) A(xchol) and (B) Cs−1(xchol) based on the three-state assumption for each of DPPC and cholesterol (details in the Supporting Information). The fitting parameters are summarized in Table S1. The simulated profile (solid curve) agrees well with the experimental data (◊) in the DPPC/cholesterol binary monolayer system at 30 mN/m except for the high xchol region. The dotted line in A and the dotted curve in B indicate the profiles calculated on the basis of ideal mixing.

indicates the presence of vicinity lipids. Therefore, we must assume that the elastic modulus of the vicinity lipid is just the same as that in the α1 state. Moreover, another type of vicinity lipid, which has a smaller elastic modulus, should be assumed because Cs−1 decreased in the xchol range from 0.1 to 0.25. Thus, three-state model cannot explain the phase behavior of the DSPC/cholesterol monolayer system, especially in the low xchol region (Figure S4 in Supporting Information). In the case of the DMPC/cholesterol monolayer system, cholesterol is added to the monolayer in the LE phase as the pure DMPC monolayer is in the LE phase irrespective of the surface pressure (Figure 4). Here, we will discuss the behavior of A(xchol) and Cs−1(xchol) in the DMPC/cholesterol monolayer system at 30 mN/m (Figure 6A) as a typical example of the effect of cholesterol on the monolayer in the LE phase. Compared to the case when cholesterol was added to monolayers in the LC phase, the Cs−1(xchol) values showed little change in the low xchol region (xchol < 0.2). However, the 9093


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interaction between deacyl PC molecules is stronger. This is consistent with the result that the condensing effect of a cholesterol molecule on the vicinity lipids, which should be reflected in the apparent partial molecular area of cholesterol in the low xchol region, was also greater as the chain length was longer (Figure 7B). On the other hand, the xchol value at the completion of CC phase formation, i.e., the value of x3, was smaller in the DPPC/cholesterol monolayer system than in the other two binary monolayer systems. We speculate that the lateral interaction between DPPC and cholesterol, not between DPPC molecules, in the CC phase may be most efficient at stabilizing the CC phase due to the good matching of the molecular length whereas the interaction between diacyl PC molecules may affect the degree of the condensing effect of cholesterol.

5. CONCLUSIONS We examined the detailed phase behaviors in the saturated diacyl PC/cholesterol binary monolayer systems by measuring π−A isotherms repeatedly under the same conditions to obtain reliable data. The intermolecular interaction between diacyl PC and cholesterol was evaluated by analyzing the cholesterol mole fraction dependence of the average molecular area A(xchol) and the area elastic modulus Cs−1(xchol) at a fixed pressure. Our analysis revealed that several phase regions can be identified and the partial molecular areas and elastic moduli of diacyl PC and cholesterol in single-phase regions can be estimated. We found that the addition of cholesterol induces a highly condensed phase (which we call the cholesterol-induced condensed (CC) phase), in which the molecular area of diacyl PC is smaller than that in the solid phase, irrespective of the hydrocarbon chain length of diacyl PC and the surface pressure. Moreover, we needed to assume at least one more state for a diacyl PC molecule other than the states in the pure diacyl PC monolayer and the CC phase in order to explain the phase behavior in the low xchol region. We demonstrated that the assumption of three states for each diacyl PC and cholesterol can sufficiently explain the behaviors of A(xchol) and Cs−1(xchol) in the DPPC/cholesterol monolayer system at 30 mN/m except for the high xchol region. Thus, the π−A isotherms with high accuracy are very useful for the analysis of the phase state in the cholesterol-containing binary monolayer system.

Figure 10. Schematic illustration of the lateral molecular packing in the DPPC/cholesterol binary monolayers in the (A) LC, (B) LC*, and (C) CC phases.

A(xchol) values deviated negatively from the ideal line, and the estimated partial molecular area of cholesterol in this region was unreasonably small (0.25 nm2). This may be due to the existence of vicinity lipids as discussed in section 4.2. If this is the case, then the behavior of Cs−1(xchol) in this region should be interpreted as the elastic modulus of the bulk and vicinity lipids being similar probably because they are insensitive to the change in surface area in the loosely packed LE phase. The formation of the CC phase was identified by the sharp increase in Cs−1(xchol) in the xchol range from 0.2 to 0.35, and the elastic modulus of diacyl PC in the CC phase was estimated to be ∼270 mN/m by curve-fitting the data at xchol = 0.35−0.5. When xchol was increased to more than 0.5, there was a breakpoint in A(xchol) at xchol ≈ 0.7 probably because of the formation of pure cholesterol domains. However, the behavior of Cs−1(xchol) in the high xchol region was not necessarily consistent with that of the A(xchol) . Further detailed study is needed to clarify the phase behavior in this region. The result of the simulation based on the above discussion is shown in the Supporting Information. In the simulation the value of x1 was set to be the same as that of x2 because there was no region assigned to the LC* phase. As expected, the calculated Cs−1 values at xchol > 0.5 deviated largely from the experimental data while the behavior in the low xchol region can be explained well by the three-state model. Finally, we discuss the dependence of the phase behavior of the diacyl PC/cholesterol monolayer system on the hydrocarbon chain length. As described in section 4.1, irrespective of the chain length, the addition of cholesterol induced the CC phase consisting of cholesterol molecules with a molecular area slightly larger than that in the pure cholesterol monolayer and diacyl PC molecules with a molecular area smaller than that in the LC phase. However, the longer the chain length, the smaller the partial molecular area of diacyl PC in the CC phase (Figure 7A). These results suggest that the molecular packing in the CC phase is tighter as the intermolecular van der Waals

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b01775. Details of the simulation based on the three-state model described in the Discussion section. Results of the application of the three-state model to the DSPC/ cholesterol system at 30 mN/m, the DPPC/cholesterol system at 10 mN/m, and the DMPC/cholesterol system at 30 mN/m. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. 9094


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ACKNOWLEDGMENTS We thank Dr. Tooru Taniguchi (Kwansei Gakuin University) for his useful discussion of the analysis of the π−A isotherm measurement. This work was partially supported by the MEXTSupported Program for the Strategic Research Foundation at Private Universities (S1201027) 2012−2016.


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